Insulin-like Growth Factor Binding Protein 4 (IGFBP4) is a member of the IGFBP family, which regulates the bioavailability and activity of insulin-like growth factors (IGFs) in various tissues. IGFBP4 binds to IGF-1 and IGF-2 with high affinity, modulating their interaction with IGF receptors and thus influencing cell proliferation, differentiation, and survival. It plays critical roles in growth, development, and metabolic processes. Unlike some other IGFBPs, IGFBP4 is unique in its ability to inhibit IGF actions unless cleaved by specific proteases like PAPP-A , which releases IGFs for receptor activation.To detect the activity of recombinant mouse IGFBP4, a functional ELISA assay was performed to evaluate the interaction between recombinant mouse IGFBP1 and recombinant chicken IGF-1.Briefly, IGFBP4 was diluted serially in PBS with 0.01% BSA (pH 7.4). Duplicate samples of 100 μl were then transferred to IGF-1-coated microtiter wells and incubated for 1h at 37℃. Wells were washed with PBST and incubated for 1h with anti-IGFBP4 pAb, then aspirated and washed 3 times. After incubation with HRP labelled secondary antibody for 1h at 37℃, wells were aspirated and washed 5 times. With the addition of substrate solution, wells were incubated 15-25 minutes at 37℃. Finally, add 50 μL stop solution to the wells and read at 450/630nm immediately. The binding activity of recombinant mouse IGFBP1 and recombinant chicken IGF-1 was shown in Figure 1, the EC50?for this effect is 0.34μg/mL.